Olga V Demakova

Russian Academy of Sciences, Moscow, Moscow, Russia

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Publications (19)67.53 Total impact

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    ABSTRACT: Drosophila melanogaster polytene chromosomes display specific banding pattern; the underlying genetic organization of this pattern has remained elusive for many years. In the present paper, we analyze 32 cytology-mapped polytene chromosome interbands. We estimated molecular locations of these interbands, described their molecular and genetic organization and demonstrate that polytene chromosome interbands contain the 5' ends of housekeeping genes. As a rule, interbands display preferential "head-to-head" orientation of genes. They are enriched for "broad" class promoters characteristic of housekeeping genes and associate with open chromatin proteins and Origin Recognition Complex (ORC) components. In two regions, 10A and 100B, coding sequences of genes whose 5'-ends reside in interbands map to constantly loosely compacted, early-replicating, so-called "grey" bands. Comparison of expression patterns of genes mapping to late-replicating dense bands vs genes whose promoter regions map to interbands shows that the former are generally tissue-specific, whereas the latter are represented by ubiquitously active genes. Analysis of RNA-seq data (modENCODE-FlyBase) indicates that transcripts from interband-mapping genes are present in most tissues and cell lines studied, across most developmental stages and upon various treatment conditions. We developed a special algorithm to computationally process protein localization data generated by the modENCODE project and show that Drosophila genome has about 5700 sites that demonstrate all the features shared by the interbands cytologically mapped to date.
    PLoS ONE 01/2014; 9(7):e101631. · 3.53 Impact Factor
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    ABSTRACT: In D. melanogaster polytene chromosomes, intercalary heterochromatin (IH) appears as large dense bands scattered in euchromatin and comprises clusters of repressed genes. IH displays distinctly low gene density, indicative of their particular regulation. Genes embedded in IH replicate late in the S phase and become underreplicated. We asked whether localization and organization of these late-replicating domains is conserved in a distinct cell type. Using published comprehensive genome-wide chromatin annotation datasets (modENCODE and others), we compared IH organization in salivary gland cells and in a Kc cell line. We first established the borders of 60 IH regions on a molecular map, these regions containing underreplicated material and encompassing ∼12% of Drosophila genome. We showed that in Kc cells repressed chromatin constituted 97% of the sequences that corresponded to IH bands. This chromatin is depleted for ORC-2 binding and largely replicates late. Differences in replication timing between the cell types analyzed are local and affect only sub-regions but never whole IH bands. As a rule such differentially replicating sub-regions display open chromatin organization, which apparently results from cell-type specific gene expression of underlying genes. We conclude that repressed chromatin organization of IH is generally conserved in polytene and non-polytene cells. Yet, IH domains do not function as transcription- and replication-regulatory units, because differences in transcription and replication between cell types are not domain-wide, rather they are restricted to small "islands" embedded in these domains. IH regions can thus be defined as a special class of domains with low gene density, which have narrow temporal expression patterns, and so displaying relatively conserved organization.
    PLoS ONE 01/2012; 7(1):e30035. · 3.53 Impact Factor
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    ABSTRACT: Salivary gland polytene chromosomes demonstrate banding pattern, genetic meaning of which is an enigma for decades. Till now it is not known how to mark the band/interband borders on physical map of DNA and structures of polytene chromosomes are not characterized in molecular and genetic terms. It is not known either similar banding pattern exists in chromosomes of regular diploid mitotically dividing nonpolytene cells. Using the newly developed approach permitting to identify the interband material and localization data of interband-specific proteins from modENCODE and other genome-wide projects, we identify physical limits of bands and interbands in small cytological region 9F13-10B3 of the X chromosome in D. melanogaster, as well as characterize their general molecular features. Our results suggests that the polytene and interphase cell line chromosomes have practically the same patterns of bands and interbands reflecting, probably, the basic principle of interphase chromosome organization. Two types of bands have been described in chromosomes, early and late-replicating, which differ in many aspects of their protein and genetic content. As appeared, origin recognition complexes are located almost totally in the interbands of chromosomes.
    PLoS ONE 01/2011; 6(10):e25960. · 3.53 Impact Factor
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    ABSTRACT: [This corrects the article on p. e25960 in vol. 6.].
    PLoS ONE 01/2011; 6(11). · 3.53 Impact Factor
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    ABSTRACT: The modern concept of intercalary heterochromatin as polytene chromosome regions exhibiting a number of specific characteristics is formulated. DNA constituting these regions is replicated late in the S period; therefore, some strands of polytene chromosomes are underrepresented; i.e., they are underreplicated. Late-replicating regions account for about 7% of the genome; genes are located there in clusters of as many as 40. In general, the gene density in the clusters is substantially lower than in the main part of the genome. Late-replicating regions have an inactivating capacity: genes incorporated into these regions as parts of transposons are inactivated with a higher probability. These regions contain a specific protein SUUR affecting the rate of replication completion.
    Russian Journal of Genetics 01/2010; 46(10):1240-1242. · 0.43 Impact Factor
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    ABSTRACT: The structural and functional analyses of heterochromatin are essential to understanding how heterochromatic genes are regulated and how centromeric chromatin is formed. Because the repetitive nature of heterochromatin hampers its genome analysis, new approaches need to be developed. Here, we describe how, in double mutants for Su(var)3-9 and SuUR genes encoding two structural proteins of heterochromatin, new banded heterochromatic segments appear in all polytene chromosomes due to the strong suppression of under-replication in pericentric regions. FISH on salivary gland polytene chromosomes from these double mutant larvae allows high resolution of heterochromatin mapping. In addition, immunostaining experiments with a set of antibodies against euchromatic and heterochromatic proteins reveal their unusual combinations in the newly appeared segments: binding patterns for HP1 and HP2 are coincident, but both are distinct from H3diMetK9 and H4triMetK20. In several regions, partial overlapping staining is observed for the proteins characteristic of active chromatin RNA Pol II, H3triMetK4, Z4, and JIL1, the boundary protein BEAF, and the heterochromatin-enriched proteins HP1, HP2, and SU(VAR)3-7. The exact cytological position of the centromere of chromosome 3 was visualized on salivary gland polytene chromosomes by using the centromeric dodeca satellite and the centromeric protein CID. This region is enriched in H3diMetK9 and H4triMetK20 but is devoid of other proteins analyzed.
    Proceedings of the National Academy of Sciences 08/2007; 104(31):12819-24. · 9.81 Impact Factor
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    ABSTRACT: In polytene chromosomes of Drosophila melanogaster, regions of pericentric heterochromatin coalesce to form a compact chromocenter and are highly underreplicated. Focusing on study of X chromosome heterochromatin, we demonstrate that loss of either SU(VAR)3-9 histone methyltransferase activity or HP1 protein differentially affects the compaction of different pericentric regions. Using a set of inversions breaking X chromosome heterochromatin in the background of the Su(var)3-9 mutations, we show that distal heterochromatin (blocks h26-h29) is the only one within the chromocenter to form a big "puff"-like structure. The "puffed" heterochromatin has not only unique morphology but also very special protein composition as well: (i) it does not bind proteins specific for active chromatin and should therefore be referred to as a pseudopuff and (ii) it strongly associates with heterochromatin-specific proteins SU(VAR)3-7 and SUUR, despite the fact that HP1 and HP2 are depleted particularly from this polytene structure. The pseudopuff completes replication earlier than when it is compacted as heterochromatin, and underreplication of some DNA sequences within the pseudopuff is strongly suppressed. So, we show that pericentric heterochromatin is heterogeneous in its requirement for SU(VAR)3-9 with respect to the establishment of the condensed state, time of replication, and DNA polytenization.
    Genetics 03/2007; 175(2):609-20. · 4.39 Impact Factor
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    ABSTRACT: In Drosophila, the dosage compensation complex (DCC) mediates upregulation of transcription from the single male X chromosome. Despite coating the polytene male X, the DCC pattern looks discontinuous and probably reflects DCC dynamic associations with genes active at a given moment of development in a salivary gland. To test this hypothesis, we compared binding patterns of the DCC and of the elongating form of RNA polymerase II (PolIIo). We found that, unlike PolIIo, the DCC demonstrates a stable banded pattern throughout larval development and escapes binding to a subset of transcriptionally active areas, including developmental puffs. Moreover, these proteins are not completely colocalized at the electron microscopy level. These data combined imply that simple recognition of PolII machinery or of general features of active chromatin is either insufficient or not involved in DCC recruitment to its targets. We propose that DCC-mediated site-specific upregulation of transcription is not the fate of all active X-linked genes in males. Additionally, we found that DCC subunit MLE associates dynamically with developmental and heat-shock-induced puffs and, surprisingly, with those developing within DCC-devoid regions of the male X, thus resembling the PolIIo pattern. These data imply that, independently of other MSL proteins, the RNA-helicase MLE might participate in general transcriptional regulation or RNA processing.
    Genetics 03/2006; 172(2):963-74. · 4.39 Impact Factor
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    ABSTRACT: Polytene chromosomes were described in 1881 and since 1934 they have served as an outstanding model for a variety of genetic experiments. Using the polytene chromosomes, numerous biological phenomena were discovered. First the polytene chromosomes served as a model of the interphase chromosomes in general. In polytene chromosomes, condensed (bands), decondensed (interbands), genetically active (puffs), and silent (pericentric and intercalary heterochromatin as well as regions subject to position effect variegation) regions were found and their features were described in detail. Analysis of the general organization of replication and transcription at the cytological level has become possible using polytene chromosomes. In studies of sequential puff formation it was found for the first time that the steroid hormone (ecdysone) exerts its action through gene activation, and that the process of gene activation upon ecdysone proceeds as a cascade. Namely on the polytene chromosomes a new phenomenon of cellular stress response (heat shock) was discovered. Subsequently chromatin boundaries (insulators) were discovered to flank the heat shock puffs. Major progress in solving the problems of dosage compensation and position effect variegation phenomena was mainly related to studies on polytene chromosomes. This review summarizes the current status of studies of polytene chromosomes and of various phenomena described using this successful model.
    International Review of Cytology 02/2004; 241:203-75. · 6.09 Impact Factor
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    ABSTRACT: In Drosophila, dosage compensation requires assembly of the Male Specific Lethal (MSL) protein complex for doubling transcription of most X-linked genes in males. The recognition of the X chromosome by the MSL complex has been suggested to include initial assembly at approximately 35 chromatin entry sites and subsequent spreading of mature complexes in cis to numerous additional sites along the chromosome. To understand this process further we examined MSL patterns in a range of wild-type and mutant backgrounds producing different amounts of MSL components. Our data support a model in which MSL complex binding to the X is directed by a hierarchy of target sites that display different affinities for the MSL proteins. Chromatin entry sites differ in their ability to provide local intensive binding of complexes to adjacent regions, and need high MSL complex titers to achieve this. We also mapped a set of definite autosomal regions (approximately 70) competent to associate with the functional MSL complex in wild-type males. Overexpression of both MSL1 and MSL2 stabilizes this binding and results in inappropriate MSL binding to the chromocenter and the 4th chromosome. Thus, wild-type MSL complex titers are critical for correct targeting to the X chromosome.
    Chromosoma 11/2003; 112(3):103-15. · 3.34 Impact Factor
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    ABSTRACT: The morphological characteristics of intercalary heterochromatin (IH) are compared with those of other types of silenced chromatin in the Drosophila melanogaster genome: pericentric heterochromatin (PH) and regions subject to position effect variegation (PEV). We conclude that IH regions in polytene chromosomes are binding sites of silencing complexes such as PcG complexes and of SuUR protein. Binding of these proteins results in the appearance of condensed chromatin and late replication of DNA, which in turn may result in DNA underreplication. IH and PH as well as regions subject to PEV have in common the condensed chromatin appearance, the localization of specific proteins, late replication, underreplication in polytene chromosomes, and ectopic pairing.
    Genetica 04/2003; 117(2-3):259-70. · 1.68 Impact Factor
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    ABSTRACT: Salivary gland polytene chromosomes of Drosophila melanogaster have a reproducible set of intercalary heterochromatin (IH) sites, characterized by late DNA replication, underreplicated DNA, breaks and frequent ectopic contacts. The SuUR mutation has been shown to suppress underreplication, and wild-type SuUR protein is found at late-replicating IH sites and in pericentric heterochromatin. Here we show that the SuUR gene influences all four IH features. The SuUR mutation leads to earlier completion of DNA replication. Using transgenic strains with two, four or six additional SuUR(+) doses (4-8xSuUR(+)) we show that wild-type SuUR is an enhancer of DNA underreplication, causing many late-replicating sites to become underreplicated. We map the underreplication sites and show that their number increases from 58 in normal strains (2xSuUR(+)) to 161 in 4-8xSuUR(+) strains. In one of these new sites (1AB) DNA polytenization decreases from 100% in the wild type to 51%-85% in the 4xSuUR (+) strain. In the 4xSuUR(+) strain, 60% of the weak points coincide with the localization of Polycomb group (PcG) proteins. At the IH region 89E1-4 (the Bithorax complex), a typical underreplication site, the degree of underreplication increases with four doses of SuUR(+) but the extent of the underreplicated region is the same as in wild type and corresponds to the region containing PcG binding sites. We conclude that the polytene chromosome regions known as IH are binding sites for SuUR protein and in many cases PcG silencing proteins. We propose that these stable silenced regions are late replicated and, in the presence of SuUR protein, become underreplicated.
    Chromosoma 04/2003; 111(6):377-98. · 3.34 Impact Factor
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    ABSTRACT: Regions of intercalary heterochromatin (IH) are dispersed in the euchromatic arms of polytene chromosomes and share the main properties of heterochromatin, namely chromosome constrictions resulting from DNA underreplication. These constrictions are frequent on the paired X chromosomes of females, but are practically absent from the single X chromosome of males. These sex-specific differences have been proposed to reflect the different levels of transcription and chromosome compaction due to dosage compensation, which in turn may affect the degree of underreplication in IH regions. To test this hypothesis, we induced dosage compensation in females by ectopic expression of MSL-2 protein. We then measured the extent of underreplication in IH regions by determining frequencies of constrictions, or by Southern blot analysis using a fragment of the ten (a) gene which is located in IH region 11A6-9. Females transheterozygous for Sxl (fhv1)/ Sxl (f1) or carrying a constitutive msl-2 transgene are known to hypertranscribe their X chromosomes. In such females, both the frequency of constrictions and DNA underreplication were reduced. Suppression of underreplication occurs only when a complete functional MSL complex assembles on the X chromosomes. We also used three strains that carried constitutive transgenes of msl-2 with mutations in the 5' untranslated regions. These strains produced normal levels of SXL protein, but variable levels of MSL-2 protein. The SXL protein did not prevent the formation of an MSL complex in these transgenic females. We found that the extent of underreplication of ten (a) DNA in IH region 11A6-9 negatively correlates with the amount of MSL complex.
    Chromosoma 08/2002; 111(2):106-13. · 3.34 Impact Factor
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    ABSTRACT: Seven new alleles of the Broad-Complex gene of Drosophila melanogaster, which encodes a family of four zinc finger protein isoforms BR-C Z1, Z2, Z3 and Z4, were generated by transposase-induced mobilization of a P[Zw] element inserted in either the first intron downstream from the P165 promoter or the exon encoding the Z2-specific zinc finger domain. They were characterized by genetic complementation tests, molecular mapping and cytogenetic analysis of their effect on ecdysone-induced puffing and BR-C proteins binding to polytene chromosomes. Four mutations that correspond to three overlapping deletions and one tandem insertion of the P[Zw] element are located in the intron. They provide evidence that regulatory elements essential for a correct expression of the BR-C Z2 and BR-C Z3 transcripts are located within the intron downstream from the P165 promoter. Three mutations correspond to internal deletions of the locus and exhibit a complete loss of all BR-C(+) genetic functions in the complementation and cytogenetic tests. They thus provide well characterized new amorphic reference alleles of the BR-C gene. The precise cytogenetic location of more than 300 binding sites of BR-C proteins on larval salivary gland polytene chromosomes was determined by immunostaining using specific antibodies. Sites were found in big ecdysone inducible puffs, constitutively active small puffs as well as interbands. A complete list of the major sites on all four salivary gland polytene chromosomes of BR-C(+) larvae is presented.
    Insect Biochemistry and Molecular Biology 03/2002; 32(2):121-32. · 3.23 Impact Factor
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    ABSTRACT: modulo belongs to the class of Drosophila genes named 'suppressor of position-effect variegation', suggesting the involvement of the encoded protein in chromatin compaction/relaxation processes. Using complementary procedures of cell fractionation, immunolocalisation on mitotic and polytene chromosomes and cross-linking/immunoprecipitation of genomic DNA targets, we have analysed the sub-nuclear distribution of Modulo. While actually associated to condensed chromatin and heterochromatin sites, the protein is also abundantly found at nucleolus. From a comparison of Modulo pattern on chromosomes of different cell types and mutant lines, we propose a model in which the nucleolus balances the Modulo protein available for chromatin compaction and PEV modification. At a molecular level, repetitive elements instead of rDNA constitute Modulo DNA targets, indicating that the protein directly contacts DNA in heterochromatin but not at the nucleolus. Consistent with a role for Modulo in nucleolus activity and protein synthesis capacity, somatic clones homozygous for a null mutation express a cell-autonomous phenotype consisting of growth alteration and short slender bristles, characteristic traits of Minute mutations, which are known to affect ribosome biogenesis. The results provide evidence suggesting that Modulo participates in distinct molecular networks in the nucleolus and heterochromatin and has distinct functions in the two compartments.
    Journal of Cell Science 10/1998; 111 ( Pt 18):2753-61. · 5.88 Impact Factor
  • O V Demakova, E S Beliaeva, I F Zhimulev
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    ABSTRACT: The position effect of the cubitus interruptus (ci) gene occurs when this gene, which is normally located in the vicinity of the pericentric heterochromatin of chromosome 4, is transferred by chromosome rearrangements to euchromatin regions. Cytological aspects of this phenomenon were investigated. For six reciprocal translocations causing the position effect (Dubinin effect) of ci, the frequencies of the ectopic contacts of the translocated chromosome 4 homologue with pericentric heterochromatin were compared to the conjugation frequencies of this chromosome's homologues. The frequencies were significantly higher when the gene was transferred to proximal chromosome regions. This suggested that the suppression of the Dubinin effect in the case of translocations with euchromatin breaks in proximal chromosome regions is caused by the higher conjugation frequency of translocated and normal chromosome 4 homologues in proximal than in distal regions. The effect of genes modulo and Su(var)2-05, which are known as modifiers of the position effect variegation, on the conjugation frequency of chromosome 4 homologues was studied for three translocations. It was shown that modulo did not affect this frequency, whereas Su(var)205 significantly decreased it. Cytogenetic data confirmed the association of the ci position effect with damage in the somatic pairing of chromosome 4 homologues. These data indicate that pericentric heterochromatin participates in determination of the localization of chromosome regions in the interphase nucleus.
    Genetika 06/1998; 34(5):629-34. · 0.37 Impact Factor
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    ABSTRACT: Classic recessive position effect variegation is related to inactivation of genes juxtaposed to heterochromatin and accompanied by cytologically visible heterochromatization (compaction) of the chromosome region containing these genes. Compaction and gene inactivation occur only in the rearranged homologue. In contrast to this, dominant variegation of the bw gene is known to involve transcriptional silencing in both the cis and trans copy, if they are paired. Our paper describes a cyto- logical approach to understanding this phenomenon. Analysis of salivary gland chromosomes carrying In(2R)bwVDe1 and In(2R)bwVDe2, evoking strong dominant bw variegation, has shown that in the rearranged homologues typical heterochromatization of the bw region and proximal neighbouring bands occurs. Heterochromatization was never observed on a normal homologue paired with a rearranged one. The insertion into the chromosome region 59E in the bwD strain is similar to pericentric heterochromatin. The insertion seems to induce heterochromatization of the neighbouring chromosome region and as a result the material of the insert and the 59E1-2 band join into a single block. When variegation is suppressed, the 59E1-2 band can be seen as a separate structure located proximal to the insert. This occurs in salivary gland polytene chromosomes of XYY males at 29 degrees C and in pseudonurse cell polytene chromosomes of otu11/otu11 females. All bands in the region of the non-rearranged homologue show normal morphology. Thus, although in all strains studied we observed heterochromatization in cis, the homologous regions in trans are not visibly affected.
    Chromosoma 08/1997; 106(2):124-32. · 3.34 Impact Factor
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    ABSTRACT: The results of the works carried out in the Laboratory of Molecular Cytogenetics (Institute of Cytology and Genetics of Siberian Branch of the RAS, Novosibirsk) devoted to the molecular genetic analysis of main units of polytene chromosomes, bands, interbands, and puffs, as well as intercalary and pericentric heterochromatin, are summarized. The results are discussed in terms of the dynamic model of organization of polytene chromosomes.
    Russian Chemical Bulletin 08/1995; 44(9):1553-1570. · 0.42 Impact Factor
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    ABSTRACT: Position-effect variegation in Drosophila melanogaster is accompanied by compaction of the corresponding chromosomal regions. The compaction can be continuous, so that bands and interbands located distal to the eu-heterochromatic junction fuse into one dense block, or discontinuous, when two or more zones of compaction are separated by morphologically and functionally normal regions. In this work it was found that in both continuous and discontinuous compaction the blocks of dense material contain the immunochemically detectable protein HP1, which has previously been characterized as specific for heterochromatin. The regions undergoing compaction do not contain HP1 when they have a normal banding pattern. Thus, it may be proposed that HP1 is one of the factors involved in compaction. If two different or two identical rearrangements are combined in the same nucleus, they variegate independently. The frequency of compaction of the two rearrangements in the same nucleus corresponds to the product of the frequencies of the compact state of the individual elements. The extent of compaction (i.e. the number of bands involved in heterochromatization) of each rearrangement does not depend on the compaction pattern of the other rearranged element.
    Chromosoma 10/1993; 102(8):583-90. · 3.34 Impact Factor

Publication Stats

299 Citations
67.53 Total Impact Points

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Institutions

  • 1998–2012
    • Russian Academy of Sciences
      • • Institute of Molecular Biology
      • • Institute of Chemical Biology and Fundamental Medicine
      • • Laboratory of Human Molecular Genetics
      Moscow, Moscow, Russia
  • 2006
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 2002
    • Institut Jacques Monod
      Lutetia Parisorum, Île-de-France, France
  • 1993
    • Institute of Cytology and Genetics
      Novo-Nikolaevsk, Novosibirsk, Russia